The long-term goal of this research is to learn more about the structure and function of herpesvirus proteins, and translate that information to new diagnostic, preventative, and therapeutic strategies for dealing with cytomegalovirus (CMV)-related diseases of man. Research proposed in this application will provide new information about the process of virion formation in herpesvirus-infected cells, and investigate two viral proteases that are of interest for their role during replication and as antiviral targets. Our focus is on three viral proteins; two that initiate and drive the earliest steps of capsid assembly, and a third that is a tegument constituent with a newly discovered ubiquitin-specific cysteine protease activity. All are essential for virus replication and we study them for the insight they provide into virus replication and host-virus interactions, and their potential for functional disruption by therapeutic agents. We study them in the context of cytomegalovirus (CMV) because this sexually transmissible agent is of increasing medical relevancy in association with AIDS, organ transplantation, cancer chemotherapy, and birth defects resulting from transplacental infections, and because CMV is the prototypic [unreadable]-herpesvirus and there is an underlying need to understand differences between it and members of the a- and ?-herpesviruses. Our four specific aims are to (1) Determine how specific interactions and conserved domains of the HCMV assembly protein precursor (pAP, pUL80.5) help it drive capsid assembly and maturation. (2) Determine the structural and enzymatic properties of the relatively uncharacterized but biologically relevant precursor form of the HCMV maturational protease precursor (pPR, pUL80a). (3) Identify features of the HCMV deubiquitinylating cysteine protease (UL48 DUB) that will lead to an understanding of its function during infection and its potential as an antiviral target. And, (4) establish high throughput assays to screen libraries for pPR and UL48 DUB inhibitors. We will apply a combination of genetic, physical, biochemical, enzymological, and biological approaches to these questions in a multidisciplinary environment that includes facilities to support this project in areas of light and electron microscopy, chemical synthesis of peptides and probes, and high throughput screens of drug and chemical compound libraries.